DK174818B1 - Polypeptide as well as the method of preparing a therapeutic drug - Google Patents

Abstract

Polypeptides consisting essentially of from about 10 to about 60 amino acid residues having alternating hydrophobic and hydrophilic amino acid residue regions are useful as pulmonary surfactants when admixed with a pharmaceutically acceptable phospholipid. The polypeptides may be represented by the formula (ZaUb)cZd, wherein: Z is a hydrophilic amino acid residue independently selected from R, D, E and K; U is a hydrophobic amino acid residue independently selected from V, I, L, C, Y and F; a has an average value of about 1 to about 5; b has an average value of about 3 to about 20; c is 1 to 10; and d is 0 to 3.y of

Description

in DK 174818 B1

The present invention relates to a polypeptide which can be used in the production of synthetic pulmonary surfactants. The polypeptide of the invention is characterized in that it consists of at least 10 amino acid residues and not more than 5 ca. 60 amino acid residues having alternating hydrophobic and hydrophilic amino acid residues represented by the formula (zaub ^ czd 'where Z means a hydrophilic amino acid residue independently selected from the group consisting of R, D, E and K, U means a hydrophobic amino acid residue independently selected from the group, which consists of V, I, L, C, Y and F, a has an average value of 1 to 5, b has an average value of 3 to 20, c is 1-10, and d is 0-3, which polypeptide, when mixed with a pharmaceutically acceptable phospholipid, forms a synthetic surfactant 15 having a surfactant activity greater than the surfactant activity of the phospholipid alone. Particularly suitable embodiments of the polypeptide are set forth in claims 2-9.

The invention also relates to a method for the preparation of a therapeutic drug which can be used for the treatment of respiratory distress syndrome, and this method is characterized by the characterizing part of claim 10.

Pulmonary surfactant (PS) feeds the alveolar epithelium of the lungs of adult mammals. Natural PS has been described as a "lipoprotein complex" because it contains both phospholipids and apoproteins, which interact to decrease the surface tension at the air-liquid interface in the lung.

Since the discovery of pulmonary surfactant and the subsequent recognition that lack thereof is the primary cause of neonatal respiratory distress syndrome (RDS), a number of studies have focused on the development of effective surfactant replacement therapy for affected individuals, especially children. exogenous PS. There are e.g. 35 improvements in lung function, as measured by a decrease in mean airway pressure and oxygen demand, have been demonstrated by the use of exogenous surfactants in preterm infants, cf. Hallman et al, Pediatrics, 21 = 473- 482 (1983);

Merritt et al., J. Pediatr., 108: 741-745 (1986); Hallman et al., J. Pediatr., 106: 963-969 (1985); Morley et al., Lancet, 5: 64-68 (1981); Merritt et al., New England J. Med., 315: 785-790 (1986); Smyth et al., Pediatrics, 71: 913-917 (1983);

Enhorning et al., Pediatrics, 76: 145-153 (1985); Fujiwara et al., The Lancet, in: 55-59 (1980); Kwong et al., Pediatrics, 26: 585-592 (1985); Shapiro et al., Pediatrics, 76: 593-599 (1985); Fujiwara in "Pulmonary Surfactant", Robertson, B.,

Van Golde, L.M.G., Batenburg J. (ed.), Elsevier Science Publishers, Amsterdam, pages 479-503, (1984).

From a pharmacological point of view, the optimal exogenous PS for use in the treatment of RDS would be one that is fully synthesized in the laboratory under controlled and sterile conditions with negligible variability in batch to batch properties. To minimize the possibility of immunological complications, the apoprotein component of an exogenous PS should be identical to that found in humans. Unfortunately, the composition of naturally occurring PS is complex and the prior art has not yet identified all the biochemical components that produce the biophysical properties necessary for high physiological activity in the lungs. In particular, it has failed in the prior art to characterize all the apoproteins present in natural PS, or to identify the function of the currently known PS apoproteins.

It should be noted that the literature on PS apoproteins and their roles in surfactant function is complex, inconsistent and sometimes contradictory, because in many studies heterogeneous apoprotein preparations have been used. Up to now, the prior art has not definitively determined the number of different apoproteins found in natural PS.

A synthetic pulmonary surfactant may comprise a pharmaceutically acceptable phospholipid admixed with a polypeptide consisting essentially of at least 10 amino acid residues and no more than ca. 60 amino acid residues corresponding in sequence to the amino acid residue sequence of human SP18 monomer, cf. the first paragraph of the description.

A method of treating respiratory distress syndrome comprises administering a therapeutically effective amount of a synthetic pulmonary surfactant comprising a pharmaceutically acceptable phospholipid admixed with an effective amount of a polypeptide which polypeptide when mixed with a pharmaceutically acceptable phospholipid. forms a synthetic surfactant with a surfactant activity greater than the surfactant activity of the phospholipid alone.

FIG. 1 shows a cDNA sequence of 750 nucleotides (top lines) and the resulting amino acid residue sequence (bottom lines). The number to the right of each line of nucleotides represents the numerical position in the nucleotide sequence at the end of each line. The nucleotides are grouped into codons, 15 codons per line, the amino acid residue encoded by each codon is shown in single letter code directly below the codon. The numerical position of certain residues in the amino acid residue sequence encoded by cDNA is shown below the residues. The amino-terminal amino acid residue of mature human SP18 monomer is Phe (encoded by nucleotides 187-189) and is designated residue # 1. The carboxy-terminal amino acid residue is Asp at residue position 81 (coded by nucleotides 427-429). Therefore, a structural gene encoding mature SP18 monomer contains 81 codons and has a nucleotide sequence corresponding to nucleotides 187-429.

FIG. 2 shows the surfactant activity of examples of polypeptide containing synthetic surfactants. The surfactant activity has been determined by measuring the pressure gradient over an air / liquid interface using the technique of pulsating bubbles. The pressure gradient (ΔP) over the surface 35 of the bubble is the absolute value of the pressure given as cm H2O. The results obtained for each synthetic synthetic surfactant are identified by the polypeptide in the surfactant. Results obtained for surfactants consisting of phospholipid alone (ie without any peptide or protein mixed therewith) have been identified as 5 PL. The results obtained using a control peptide having only 8 amino acid residues and having a sequence corresponding to residues 74-81 in human SP18 monomer are also shown (p74-81). The data points shown are obtained after 15 seconds, 1 minute and 5 minutes.

FIG. 3 shows a series of two curves illustrating the results of a static compliance study of examples of synthetic surfactants using the rabbit fetal model previously described by Revak et al, Am. Rev. Respir. Dis., 134: 1258-1265 (1986). After instillation of a synthetic surfactant or control in the trachea, the rabbit is given artificial respiration for 30 minutes before static compliance measurements are taken. The "x" axis shows the pressure in cm of water, while the "y" axis shows the volume in ml / kg of body weight.

The curve to the left shows values at inflation, and the curve 20 to the right shows pumping values. The results for the following surfactants tested are shown: naturally occurring surfactant (open square with a dot in the middle), phospholipid (PL) with 7% p52-81 (a polypeptide corresponding to residues 52-81 in SP18) (filled panes), PL with 3% p52-81 (filled 25 squares with white dot in the middle), PL with 7% p36-81 (open panes), PL with 3% p66-81 (filled squares), PL with 3% pi-15 (open squares) and PL control (filled triangles).

FIG. 4 shows a series of two curves showing the results of a static compliance study of Examples 30 of synthetic surfactants. The method performed is as described for FIG. 7, except that another method of drip is used. The "x" and "y" axes and curves to the left and right are as described for Fig. 7. The results for the following surfactants tested are shown: naturally occurring surfactant (open squares with a dot in the middle), phospholipid (PL) with 10% p51-81 (closed panes), 5 DK 174818 B1 PL with 10% p51-76 (filled squares) and PL (filled triangles).

A. Definitions.

5 Amino Acid: All amino acid residues identified here are in the naturally occurring L configuration. In accordance with standard polypeptide nomenclature, J. Biol. Chem., 243: 3557-3559 (1969), are abbreviations for amino acid residues as shown in the following correspondence table: 10 Correspondence Table

Symbol Amino acid l letter 3 letters 15 Y Tyr L-tyrosine G Gly glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine 20 S Ser L-serine I Ile L-isoleucine L Leu L-leucine T Thr L- threonine V Val L-valine 25 P Pro L-proline K Lys L-lysine H His L-histidine Q Gin L-glutamine E Glu L-glutamic acid 30 W Trp L-tryptophan R Arg L-arginine D Asp L-aspartic acid N Asn L-asparagine C Cys L-cysteine 35 -

It should be noted that all of the amino acid residue sequences herein are represented by formulas whose orientation from left to right is in the conventional direction from the amino terminus to the carboxy terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a bond to a group such as H and OH (hydrogen and hydroxyl) at 5 and 5, respectively. amino and carboxy terminus or an additional sequence of one or more amino acid residues. In addition, it should be noted that a slash at the right end of a residual sequence indicates that the sequence is continued on the next line.

Polypeptide and Peptide: Polypeptide and Peptide are terms which are used interchangeably here to denote a linear sequence of not more than ca. 60 amino acid residues linked to each other by peptide bonds between the α-amino and carboxy groups in adjacent residues.

Protein: Protein is a term that is used here to denote a linear sequence of over approx. 60 amino acid residues linked to each other as in a polypeptide.

Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate and a nitrogen-containing heterocyclic base.

The base is bonded to the sugar moiety via the glycoside carbon atom (1 'carbon in the pentose), and this combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bound to the 3 'or 51 position of the pentose, it is referred to as a nucleotide.

Base pair (bp): A partnership between adenine (A) and thymine (T) or between cytosine (C) and guanine (G) in a double-stranded DNA molecule.

"Surfactant activity" for a polypeptide is defined as the ability, in combination with lipids, either alone or in combination with other proteins, to exhibit activity in the in vivo assay according to Robertson, Lung, 158: 57-68 (1980). In this test, the sample to be evaluated is administered through an endotracheal tube to rabbit fetuses or to lambs born prematurely by cesarean section. These "time-35 corpses" lack their own PS and are supported with artificial respiration. Measurements of pulmonary remission, blood gases and B1 fan pressure provide indications for activity. Prior assessment of activity can also be done by an in vitro assay, e.g. as described by King et al.

Am. J. Physiol., 223: 715-726 (1972), or the one illustrated below, which utilizes a surface tension measurement at an air / water interface when a polypeptide is mixed with a phospholipid.

B. Polypoeotides.

A polypeptide of the invention (an object of the polypeptide) is characterized by its amino acid residue sequence and its novel functional properties. When blended with a pharmaceutically acceptable phospholipid, the subject polypeptide forms a synthetic pulmonary surfactant having a surfactant activity greater than the surfactant activity of the phospholipid alone (as shown by a lower ΔΡ as shown in Fig. 2 and a higher volume per given pressure as shown in FIG.

3 and 4).

In another embodiment, a polypeptide of the invention has an amino acid residue sequence having a compound hydrophobicity of less than zero, preferably below or equal to -1, especially below or equal to -2. The determination of the composite hydrophobicity number of a peptide is described in detail in Example 1. These hydrophobic polypeptides perform the function of the hydrophobic region of SP18. In a preferred embodiment, the amino acid sequence mimics the pattern of hydrophobic and hydrophilic residues in SP18.

A preferred hydrophobic polypeptide comprises a sequence of alternating hydrophobic and hydrophilic amino acid residues -30 regions and is characterized as containing at least 10 amino acid residues represented by the formula (ZaUb) czd · Z and U are amino acid residues such that Z and U are independently selected at each occurrence. Z is a hydrophilic 35 amino acid residue selected from the group consisting of R, D, E and K. U is a hydrophobic amino acid residue selected from the group consisting of V, I, L, C, Y and F. "a", "b", "c" and "d" are numbers indicating the number of hydrophilic or hydrophobic residues, "a" having an average value of from 1 to 5, preferably from 1 to 3. " b "has an average value of 5 from 3 to 20, preferably from 3 to 12, especially from 3 to 10. nc" is 1-10, preferably 2-10, especially 3-6. "d" is 0-3, for incremental 1 or 2.

When indicated that the amino acid residue represented by Z and U is independently selected, it is believed that at each occurrence, a residue is selected from the specified group. When "a" e.g. is 2, that is, each of the hydrophilic residues represented Z will be independently selected and thus e.g. may include RR, RD, RE, RK, DR, DD, DE, DK. When indicated that "a" and "b" have average values, this means that the average values of Ma "and" b ", although the number of residues in the repetitive sequence (Za, Uj-j) may vary somewhat in the peptide sequence, would be from about 1 to about 5 and from about 3 to about 20, respectively.

Examples of preferred polypeptides of the above formula are shown in Table III.

Table III

Designation · * · The amino acid residue sequence 25 DL4 DLLLLDLLLLDLLLLDLLLLD, RL4 RLLLLRLLLLRLLLLRLLLLR, RL8 RLLLLLLLLRLLLLLLLLRLL, RL7 RRLLLLLLLRRLLLLLLLRRL, RCL1 RLLLLCLLLRLLLLCLLLR, 30 RCL2 RLLLLCLLLRLLLLCLLLRLL, RCL3 RLLLLCLLLRLLLLCLLLRLLLLCLLLR. 1 35

The term is an abbreviation for the specified amino acid residue sequence.

9 DK 174818 B1

Also included are compound polypeptides having from 10 to 60 amino acid residues. A composite polypeptide consists essentially of an amino-terminal sequence and a carboxy-terminal sequence. The amino terminal sequence has an amino acid sequence from a hydrophobic region of a polypeptide or hydrophobic peptide of the invention, preferably a hydrophobic polypeptide, as defined in the above formula. The carboxy-terminal sequence has the amino acid residue sequence of a subject carboxy-terminal peptide.

A polypeptide of the invention can be synthesized by any technique known to those skilled in the art of polypeptide. An excellent overview of the many types of technique available can be found in J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co, San Fran-cisco, 1969, and J. Meienhofer, "Hormonal Proteins and Peptides", Volume 2, page 46, Academic Press (New York), 1983, for solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", Volume 1, Academic Press (New York), 1965, for classical solution synthesis.

In general, these methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid residue is protected by a suitable selectively removable protecting group. Another selective removable protecting group is used for amino acids containing a reactive side group such as lysine.

Using a solid phase synthesis as an example, the protected or derivatized amino acid is linked to an inert solid carrier through its unprotected carboxyl or amino group. Then, the protecting group on the amino or carboxyl group is selectively removed and the next amino acid in the sequence having the complementary group (amino or carboxyl) suitably protected is admixed and reacted under conditions suitable to form the amide bond with the residue already present. is attached to the solid carrier. Then, the protecting group on the amino or carboxyl group is removed from this newly added amino acid residue, and then the next amino acid (appropriately protected) is added, etc. After all the desired amino acids have been linked in the correct sequence, any remaining terminal and side protecting groups (and the solid carrier) sequentially or simultaneously to obtain the final polypeptide.

C. Synthetic surfactants.

A present polypeptide may be admixed with a pharmaceutically acceptable phospholipid to form a synthetic pulmonary surfactant (PS) which can be used in the treatment of respiratory distress syndrome.

The term "pharmaceutically acceptable" refers to 15 molecular moieties and materials which do not produce an allergic or similar adverse reaction when administered to a human.

Phospholipids which can be used to form synthetic alveolar surfactants in admixture with protein are well known in the art. Notter et al., Clin. Perinatology, 14,433-479 (1987), has an overview of the use of both native and synthetic phospholipids for synthetic surfactants.

A synthetic pulmonary surfactant effective in treating RDS comprises an effective amount of a treated polypeptide mixed with a pharmaceutically acceptable phospholipid. While methods for determining the optimal polypeptide: phospholipid weight ratios for a given polypeptide-phospholipid combination are well known, therapeutically effective amount ratios are in the range of about 1: 5 to approx. 1: 10 to 30,000, preferably from ca. 1: 100 to approx. 1: 5000 and especially from approx. 1: 500 to approx. 1: 1000. In a more preferred embodiment, the polypeptide: phospholipid weight ratio is in the range of about 1: 5 to approx. 1: 2000, preferably from ca. 1: 7 to approx.

1: 1000 and especially from approx. 1:10 to approx. 1: 100. Thus, a synthetic pulmonary surfactant according to the invention may contain from ca. 50, usually from ca. 80, to almost 100 wt.% Of lipid and from ca. 50, usually from ca. 20, to less than 1% by weight polypeptide. Preferably, a subject polypeptide of 1 to approx. 10% by weight of the surfactant for polypeptides corresponding to portions of the SP18 sequence and 1: 100 for polypeptides 5 corresponding to the entire SP18 monomer.

The lipid moiety is preferably from ca. 50 to approx. 90, preferably from ca. 50 to approx. 75, wt% dipalmitoylphosphatidylcholine (DPPC), the remainder being unsaturated phosphatidylcholine, phosphatidylglycerol (PG), triacylglycerols, palmitic acid sphingomyelin or mixtures thereof. The synthetic pulmonary surfactant is prepared by mixing a solution of a subject polypeptide with a suspension of liposomes or by mixing the subject polypeptide and lipids directly in the presence of an organic solvent. Then, the solvent is removed by dialysis or evaporation under nitrogen and / or subjected to vacuum.

The present synthetic pulmonary surfactant is preferably formulated for endotracheal administration, i.e. typically as a liquid suspension, as a dry powder "powder" or as an aerosol. Eg. For example, a synthetic surfactant (polypeptide-lipid micelle) is suspended in a liquid with a pharmaceutically acceptable excipient, e.g. water, saline, dextrose or glycerol. A surfactant-containing therapeutic composition may also contain small amounts of non-toxic excipients such as pH-value buffers, including e.g. sodium acetate and sodium phosphate. To prepare a synthetic surfactant in powder form, a synthetic surfactant is prepared as described herein, after which it is lyophilized and recovered as a dry powder.

If it is to be used in aerosol administration, the present synthetic surfactant is supplied in finely divided form with an additional surfactant and propellant. Typical surfactants which may be administered are fatty acids and their esters.

However, in the present case, it is preferred to use other components of the surfactant complex DPPC and PG. Useful propellants are typically gases under ambient conditions and condensed under pressure. Lower alkanes and fluorinated alkanes such as Freon can be used. The aerosol is packed in a container equipped with a suitable valve so that the ingredients can be kept under pressure until released.

A synthetic surfactant is administered, as is appropriate according to the dosage form, via an endotracheal tube, by aerosol administration or by atomization of the suspension or powder into the inhaled gas. Amounts of synthetic PS of between approx. 1.0 and approx. 400 mg / kg, preferably 10 from ca. 50 mg / kg to approx. 500 mg / kg, administered at a dose.

For use in newborn infants 1-3, administration is usually sufficient. In adults, sufficiently reconstituted complex is administered to produce a PO2 in the normal range (Hallman et al., J. Clinical Investigation, 70: 673-15682 (1982)).

The following examples are intended to illustrate the present invention.

Examples Example 1

In vitro assessment of polypeptide surfactant activity A. Methods.

Measurement of surfactant activity.

Measurements of surface pressure over an air-liquid interface (expressed in negative cm water pressure) at minimal bubble radius have been determined at various times using the pulsed bubble technique described by Enhorning, J. Appl. Physiol., 43:198-203 (1977).

Briefly, the Enhorning surfactometer (Surface 30 International, Toronoto, Ontario) measures the pressure gradient (ΔΡ) over a liquid-air interface in a bubble that pulses at a rate of 20 cycles / minute between a maximum (0.55 mm ) and a minimal (0.4 mm) radius. Bubbling, which forms in a 37 ° C hot, water-enclosed sample chamber of 20 μΐ, is monitored by microscopic optics while the pressure changes are recorded on a recorder strip card calibrated for 13 DK 174818 B1 0 and -2 cm H2 O.

Determination of the composite hydrophobicity figure.

The composite hydrophobicity number for each peptide is determined by assigning each amino acid residue in a peptide its corresponding hydrophilicity number as described in Table 1 of Hopp et al, Proc. Natl. Acad. Sci., USA, 7,8: 3824-3829 (1981), to which reference is made herein.

For a given peptide, the hydrophilicity numbers are summed and the sum represents the composite hydrophobicity number.

Preparation of Synthetic Surfactants.

After mixing with solvent, each peptide is combined with phospholipids (DPPC: PG, 3: 1) to form a synthetic surfactant according to one of the following methods.

Method A is carried out by mixing 16 μΐ peptide / solvent mixture (40 μg peptide) with 100 μΐ chloroform containing 400 µg phospholipids, stirring the mixture for approx. 10 minutes at 37 ° C to form a peptide / phospholipid mixture. Chloroform is removed from the peptide / phospholipid mixture by drying under N 2. The synthetic surfactant thus formed is then mixed with 90 μΐ H2 O and stirred gently for approx. 10 minutes at 37 ° C. Then mix 10 μΐ 9% NaCl in the surfactant-containing solution.

Method B is carried out by first placing 100 μΐ of chloroform containing 400 μg of phospholipids in a glass tube and removing the chloroform by drying under N2 for approx. 10 minutes at 37 ° C. 16 μΐ peptide / solvent mixture and 74 μΐ H2 O are mixed with the dried phospholipids, after which 30 is stirred gently for approx. 10 minutes at 37 ° C. To the synthetic surfactant thus formed, 10 μΐ 9% NaCl is mixed.

Method C is carried out by first holding the polypeptide-PL mixture at 43 ° C for 10 minutes, after which time the solvents are removed from the mixture by drying under 35 N 2. When necessary, the mixtures are further dried by subjecting them to a vacuum for 15 minutes to give a dried polypeptide-PL mixture. Thereafter, water is mixed with each dried mixture in an amount intended to equal 90% of the volume needed to achieve a final PL concentration of either 4 or 5 10 mg / ml (as shown in Table VII), to form another mixture. This second mixture is kept for 1 hour at 43 ° C with stirring. Next, a volume of 6% NaCl equal to 10% of the volume needed to achieve the desired PL concentration is mixed with the second mixture and the resulting final mixture is maintained for 10 minutes at 43 ° C. .

In most cases, the final mixture is subjected to a final step of 3 cycles of freezing and thawing.

B. Results.

The synthetic surfactants shown in Table VII 15 have been prepared as shown in the Table.

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Each of the synthetic surfactants listed in Table VII has been tested for surfactant activity as demonstrated by their in vitro surface tension reducing ability using the "Enhorning" bubble test as described above.

The results of this study, shown in Figs. 3, shows that a subject polypeptide, when mixed with pharmaceutically acceptable phospholipids, forms a synthetic pulmonary surfactant which has greater surfactant activity than the phospholipids alone, as shown by the lower ΔΡ values. Typically, using the polypeptides ΛΡ values which are 10-80% lower are obtained. It should be noted that the control peptide p74-81 of 8 amino acid residues which fall outside the scope of the present invention does not form a synthetic PS with greater activity than the phospholipid alone, indicating that the amino acid residue length is a critical feature.

The surfactant activity of further examples of polypeptides of the invention has been studied using the "bubble testing" described above. The results of this study are shown below in Table VIII.

Each polypeptide has been mixed with the indicated solvent at a concentration of 2.5 mg polypeptide per ml. ml of solvent. The resulting mixture 25 has been observed to determine whether a solution or suspension of insoluble polypeptide is formed. The mixtures that form a suspension are further mixed by water bath sonication for 10 seconds to form a very fine suspension sufficient for pipetting using glass pipettes.

After the solvent mixture, each peptide is mixed with phospholipids (PL), DPPC: PG, 3: 1, in chloroform in a glass tube, so that the amount of polypeptide added is equal to one tenth (10% by weight) of the amount of PL added. to form a synthetic surfactant according to either method A, B or C.

17 DK 174818 B1

Thereafter, each of the synthetic surfactants for surfactant activity is tested as shown by their ability to reduce the surface tension in vitro by a bubble test conducted as described above. The pressure gradient (5 UP) is a measure of surfactant activity in the polypeptide PL final mixture and it is determined using an Enhorning surfactometer as described above. Measurements are obtained at times 15 seconds? 1 minute and 5 minutes and is expressed as an average of three independent measurements for the 10 polypeptide-PL mixture indicated. Pressure gradient measurements for comparative samples of PL alone {PL) and naturally occurring human surfactants have been determined as controls.

The results of this study are shown in Table VIII.

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(1) cn CD CD r-XroX X & U O U X <0 a a a a a 0-- tx κ oi & & ai a s 20 DK 174818 B1 (1) It is stated whether the initial mixture of peptide and solvent is a solution or suspension.

(2) The letters indicate the method used to prepare the synthetic surfactant. These methods are described above. A "+" indicates that the final mixture has been subjected to a final step of 3 cycles of freezing and thawing. Another indicates that this step has not been completed.

(3) Concentration ("Kone.") Of phospholipid (PL) in the final mixture is given in mg PL per ml. ml of mixture.

(4) The pressure gradient is a measure of surfactant activity in the polypeptide PL final mixture as determined using an Enhorning surfactometer as described above. Measurements were obtained at three time points of 15 seconds (15s), 1 minute (1m) and 5 minutes (5m) and are expressed as an average of three independent measurements of the polypeptide-PL mixture indicated. Pressure gradient measurements for comparative samples of PL alone 20 (PL) and naturally occurring human surfactant are also shown.

(5) These solutions have been prepared at a concentration of 20 mg / ml PL and have been diluted to 10 mg / ml prior to testing.

25

These results indicate that, when mixed with pharmaceutically acceptable phospholipids, a subject polypeptide forms a synthetic pulmonary surfactant which has greater surfactant activity than the phospholipids alone, as shown by the higher volume per given pressure.

21 DK 174818 B1

Example 2

In vivo assessment of synthetic surfactant activity.

A. Methods.

Preparation of Synthetic Surfactants.

A subject polypeptide is first mixed with solvent as described in Example 1. The resulting mixture is further mixed with phospholipid (PL) such that the amount of polypeptide added is either 3%, 7% or 10% by weight of the amount of PL added. shown below. The polypeptide-10-PL final mixture (synthetic surfactant) is formed by Method C using the final freeze-thawing step as detailed in the section ("Preparation of Synthetic Surfactants" in Example 2, except that the final mixture has a concentration of 20 mg phospholipid per ml final mixture.

15

Tildrvpningsprotokoler.

Protocol 1: Rabbit fetuses are treated by injection into the trachea of a 0.1 ml solution containing either a synthetic surfactant prepared in Example 3A or 2 mg of 20 native surfactant (NS) prepared as described in Example 1 or 2 mg PL.

Protocol 2: Synthetic surfactant is drip into rabbit-fetal lung by injection into trachea from a single syringe of the following three components, so that the components come out of the syringe in the following order: (1) 0.05 ml of air, (2) 0.1 ml of a synthetic surfactant prepared in Example 3A or 2 mg of PL or 2 mg of native surfactant and (3) 0.1 ml of air.

Protocol 3; From one syringe, a 0.1 ml portion prepared as described in Example 3A (or 2 mg 30 NS or PL) in the rabbit trachea as described above is injected, followed by injection of 0.05 ml of lactated Ringer's solution and 0.2 ml of air from a syringe. second syringe.

Protocol 4: From one syringe, 0.1 ml of a synthetic surfactant prepared as described in Example 35 3A (or 2 mg NS or PL), 0.15 ml of air, 0.1 ml of saline solution and 0.3 ml of air are injected into the trachea. as described above. Two subsequent portions of 0.3 ml of air are given.

Protocol 5: Rabbit fetuses are treated by injection into the trachea from a single syringe with the following four components, so that the four components emerge from the syringe by 5 injection in the order indicated: (1) 0.2 ml solution containing either a synthetic surfactant prepared in Example 3A or 4 mg of native surfactant or 4 mg of PL, (2) a volume of 0.15 ml of air, (3) 0.1 ml of N saline solution and (4) a volume of 0.3 ml of air. The above injection is repeated 10 15 minutes after the first injection.

Protocol 6: Rabbits are treated as described in Protocol 5 except that the two subsequent portions of 0.3 ml of air are given after the first injection and no further injection is given after 15 minutes.

15

Rabbit fetal model for studying surfactant activity.

The surfactant activity of examples of polypeptides of the invention has been studied using the methods previously described in detail by Revak et al., Am. Rev. Respir. Dis., 134: 1258-1265 (1986), with the exceptions set forth below.

Rabbit embryos, which are 27 days into gestation, are removed by hysterotomy and immediately injected with 0.05 ml of "Norcuron®" (Organon, Inc., NJ) to prevent spontaneous breathing. The rabbit embryos are then weighed and a small cannula is inserted into the trachea by tracheotomy. Synthetic surfactants prepared as described above are then drip into the rabbit fetal lung by one of the above drip protocols.

After drip, the rabbit is placed in a specially designed plethysmograph (containing a Celesco transducer) connected to a fan (Baby Bird, Baby Bird Corp.,

Palm Springs, CA), and the treated lung is ventilated at a rate of 30 cycles per per minute with a maximum inhalation pressure of 25 cm H2O, a positive final exhalation pressure of 35 4 cm H2O and a respiration time of 0.5 seconds. In some studies, dynamic compliance measures are taken at different times during the respiratory procedure. In others, measurements of static indulgence are made after artificial respiration.

Static compliance measurements are taken after 30 5 minutes of artificial respiration. The animals are removed from the ventilator and the lungs are degassed at -20 cm H 2 O in a bell under vacuum. Then the lungs are first pumped up and then pumped out through a tee which is connected to a tracheostomy tube.

The volume of air needed to obtain static 10 pressures of 5, 10, 15, 20, 25, and 30 cm of H 2 O is measured during both the inflating and exhaust phases to generate static pressure / volume curves as a measure of static compliance. .

Using the plethysmograph, measurements of dynamic compliance are made at various times during a period of artificial respiration of 60 minutes. Computer-aided data analysis results in compliance data expressed as ml of air per day. cm H 2 g body weight at each individual time. Compliance is calculated using the formula below.

Affection = AV

ΔΡ APtp = (C) -1 · (AV) + (R) · (F) 25 Ptp = transpulmonary pressure C = compliance (elastic component - relates change in volume to pressure) 30 R = resistance (relates flow to pressure) F = flow 3 5 V = volume = integral of flow over time

The above equation is solved by a multiple linear regression for C and R. The indulgence (C) represents the elastic nature of the lungs and the resistance (R) represents the pressure needed to overcome the resistance against the current. of gas into and out of the lungs.

B. Results.

Static compliance data using the 5 drip protocols 1 and 5 are shown in Figs. FIG. 7 and FIG. 8. Improved pulmonary remission is seen in all lungs treated with naturally occurring surfactant or with the tested synthetic surfactants compared to the lungs treated with phospholipids (PL) alone, with one exception. The synthetic surfactant produced using pL-15 (Fig. 7) does not produce improved pulmonary compliance with PL alone as measured by static compliance.

The results of the studies of dynamic compliance 15 are shown in Table IX.

TABLE IX.

DK 174818 B1

Dynamic yield in ml air / cm HnO x 104 5 g body weight%

peptide

Together- Minutes after Sample similar to surfactant drip_ given at 10 with PL 10 20 30 40 50 60 protocol no.

PL

7 8 7 10 11 15 4 24 22 23 23 22 20 4 15 15 16 17 18 21 29 4

NS

265 261 168 186 173 147 * 4 418 388 405 288 237 * 4 20 155 176 172 172 179 * 4 P36-81 5% 255 146 * 3 5% 245 291 3 25 10% 154 1,162 2 10% 252 623 2 P44- 80 10% 1 27 87 138 207 323 6 30 10% 1 20 23 35 59 87 136 6 P51-80 10% 1 42 114 247 300 6 10% 1 6 35 - P52-81 5% 517 226 * 3 5% 434 55 * 3 10% 195 1,243 2 40 10% 43 1,690 2 TABLE IX (continued) 26 DK 174818 B1

Dynamic compliance in ml air / cm HnO x 104 5 9 body weight%

peptide

Together- Minutes after Sample similar to surfactant drip given by 10 with PL 10 20 30 40 50 60 protocol no.

P51-76 10% 33 22 56 87 124 85 4 15 10% 10 11 186 358 141 144 * 4 10% 15 36 109 241 264 301 4 P51-E0 10% 17 41 52 78 99 208 4 20 10% 76 94 149 149 217 308 4 10% 23 71 130 156 182 109 * 4 10% 42 114 247 388 6 P59-80 25 10% 1 24 34 68 107 146 132 6 10% 1 55 111 190 300 376 6 p64- 80 10% 1 63 129 235 318 6 3 0 - 1 Before dripping to the rabbits, these samples are filtered through a 25 μπ \ filter.

35 * A decrease in compliance with time may indicate the development of pneumothorax.

As shown in Table IX, each of the synthetic surfactants of the invention and naturally occurring surfactant improve dynamic compliance values compared to phospholipid alone.

C. Discussion.

The in vivo compliance studies show that the use of a number of exemplary synthetic surfactants of the invention results in increased compliance compared to phospholipid alone for each of the synthetic surfactants tested. Therefore, when mixed with pharmaceutically acceptable phospholipids, the proteins and polypeptides of the invention form synthetic surfactants which have greater surfactant activity than phospholipid alone. Use of the synthetic surfactants is advantageous for generating improved compliance values in vivo.

5

Example 3

Study of binding of C-terminal peptide to lung epithelial cells.

A. Methods.

10 Peptide Binding Assay.

A peptide with residues 74-80 in SP18 (VLRCSMD) is radiolabeled by the Bolton-Hunter method (Bolton et al, Biochem J., 133: 529-538 (1973)) with 125 I (New England Nuclear - 34.1 moles). / ml, 28.0 ng / ml, 75 μΟί / τηΙ).

Human pulmonary epithelial cells (human lung carcinoma cell, ATCC reference no. CCL 185, commonly known as A549 cells) are grown to confluence in 6-culture tissue culture dishes. In this study, the following solutions are used: 20 PBS / BSA: 10 mM Na-phosphate + 0.15 M NaCl + 0.5% BSA, pH 7.4.

Light buffer: 1% SDS in water.

Solution F: 5 ml PBS / BSA + 51.56 μg cold peptide.

Solution D: 2.5 ml PBS / BSA + 87 µl - ^^ I peptide.

Solution D 1/5: 0.5 ml D + 2.0 ml PBS / BSA.

Solution D 1/25: 0.5 ml D 1/5 + 2.0 ml PBS / BSA.

Solution E: 2.5 ml PBS / BSA + 87 μΐ 125 I peptide 35 + 20.78 μg cold peptide.

Solution E 1/5: 0.5 ml E + 2.0 ml PBS / BSA.

Solution E 1/25: 0.5 ml E 1/5 + 2.0 ml PBS / BSA.

Three 6-hole plates are pretreated by incubating with 0.5 ml of the solutions below for 15 minutes at 22 ° C. The holes with odd numbers are pretreated with PBS / BSA, and the holes with even numbers are pretreated with solution F. After removal of the pretreatment solution, the holes are incubated with 0.5 ml of the following solutions at 22 ° C for the specified time while the plates cradled slightly.

5

Hole Sample Incubation Time ID 7 minutes 2 E 7 minutes 3 D 1/5 7 minutes 10 4 E 1/5 7 minutes 5 D 1/25 7 minutes 6 E 1/25 7 minutes 7 D 30 minutes 15 8 E 30 minutes 9 D 1/5 30 minutes 10 E 1/5 30 minutes 11 D 1/25 30 minutes 12 E 1/25 30 minutes 20 13 D 143 minutes 14 E 143 minutes 15 D 1/5 143 minutes 16 E 1/5 143 minutes 25 17 D 1/25 143 minutes 18 E 1/25 143 minutes

At the end of the incubation time, the above liquid is removed from each hole and stored for 30 count. Each hole is washed four times with cold (4 ° C) PBS / BSA. The washing fluids are stored for counting. The plate is then brought back to room temperature and 1 ml of light buffer is added to each hole. The plate shakes slightly until all cells are lysed and detached from the plate (3-4 minutes -35 hours). The solution is removed from each hole and counted.

Another 1 ml of light buffer is added to each hole, mixed for a few minutes and removed for counting bound counts. The percentage and absolute amount of counts bound are determined.

Specific counts are determined by subtracting the counts 5 bound in holes containing unlabelled (cold) peptide from the corresponding hole without cold peptide. The results are shown in Table X below.

The method is repeated with the following changes:

Dx = 1433 μΐ PBS / BSA + 167 μΐ l25I peptide [1.78 pmol / 500 10 μΐ].

D2 = 183.3 μΐ PBS / BSA + 366.7 μΐ Dx [1.19 pmol / 500 μΐ].

D3 = 275 μΐ PBS / BSA + 275 μΐ D] _ [0.89 pmol / 500 μΐ].

D4 = 366.7 μΐ PBS / BSA + 183.3 μΐ Dx [0.59 pmol / 500 μΐ].

Ds = 458.3 μΐ PBS / BSA + 91.7 μΐ Dx [0.30 pmol / 500 μΐ].

D6 = 513.3 μΐ PBS / BSA + 36.7 μΐ Dx [0.12 pmol / 500 μΐ].

E1 = 1386.24 μΐ PBS / BSA + 167 μΐ 125I peptide [4.676 ng] + 46.76 μΐ cold peptide at 100 μg / ml [4.676 μg).

E2-Eg Diluted as above for D2 - Dg.

F = 3.398 ml PBS / BSA + 102.29 μΐ cold peptide at 20 100 μg / ml.

Two 6-hole plates are washed once with 1 ml PBS / -BSA. The holes with odd numbers are pre-treated with PBS / BSA and the holes with even numbers are pre-treated with solution F. After 25 removal of the pre-treatment solution, the following solutions are added:

Hollow Sample Hollow Sample 1 Dx 7 D4 2 Ex 8 E4 30 3 D2 9 D5 4 E2 10 Oak 5 D3 11 Dg 6 E3 12 Oak 3 5 The solutions are incubated for 30 minutes at room temperature with gentle wetting. Then the above liquids are removed and stored for counting. Each hole is washed four times with 0.5 ml of cold PBS / BSA. Washing liquids are stored for counting.

1 ml of 1% SDS is added to each hole to make the cells soluble. After 3 minutes, it can be seen that all 5 cells are detached from the plate. The lysed cell-containing supernatant is counted along with another SDS wash of the holes. Total counts and the percentage of bound counts are determined. Specific binding is determined by subtracting the counts bound in holes containing cold peptide from the corresponding hole without cold peptide. The results are shown in Table XI.

B. Results.

The results of the binding studies are shown in Tables X and XI below.

TABLE X

31 DK 174818 B1

Total Specific CPM% Counts 5 Hole Total CPM Bound Bound Difference Bound1 1 1,109,126 24,414 2.23 2 1,087,659 17,353 1.60 0.63% 6,930 10 3 223,170 4,479 2,01 4,221,608 4,113 1.86 0.15 % 330 5 45,877 828 1.80 6 47,731 880 1.84 -0.04% - 18 15 7 1,103,606 25,905 2,35 8 1,152,287 19,230 1,67 0,68% 7,480 9 227,396 4,996 2.20 20 10 230,974 4.137 1.79 0.41% 901 11 47.899 1.030 2.15 12 49.894 922 1.85 0.30% 132 25 13 1.151.347 10.071 0.87 14 1.108.755 9.506 0.86 0.01% 110 15 220,340 1,692 0.77 16 229.253 1.800 0.79 -0.02% - 44 30 17 46.893 407 0.87 18 47.426 386 0.81 0.06% 26

Corrected to 1,100,000 cpm / undiluted tube.

TABLE XI

32 DK 174818 B1

Total CPM% Corrected 5 Hole Total CPM Bound Bound Difference pmol 1 3,070,705 66-954 2.78 2 2.995.775 56.055 1.87 9.390 1.78 10 3 2.029.323 39.562 1.95 4 2.013.557 33.573 1, 67 5,723 1,19 5 1,436,189 26,883 1,87 6 1,427,731 25,073 1,76 1,755 0,89 15 7 994,288 15,669 1,58 8 964,481 14,776 1,53 503 0,59 9 460,317 6-513 1.41 20 10 479.746 6.816 1.42 - 52 0.30 11 202.494 2.930 1.45 12 192.990 2-806 1.45 0 0.12 25 * Corrected for 1.78 pmol = 3,033,740 cpm.

C. Discussion.

As can be seen from the data in Table X, the study has shown that the peptide is binding specific to the cells as shown by competing inhibition of unlabeled peptide. However, the cells have not been saturated with the amount of labeled peptide used in this study. In addition, the peptide decomposes after 143 minutes.

The second study was carried out using the 30-min incubation period and an increased amount of labeled peptide to obtain saturation of the cells. As will be seen in Table XI, specific binding has again been demonstrated. Furthermore, saturation has been achieved as shown by flattening the amount of bound counts at high concentration of labeled peptide.

Thus, the binding studies show that the C-terminal peptide of the invention binds specifically to lung epithelial cells.

Claims (10)

33 DK 174818 B1 PATENT REQUIREMENT · 1. A polypeptide, characterized in that it consists of at least 10 amino acid residues and not more than ca. GO amino acid residues with alternating hydrophobic and hydrophilic amino acid residues represented by the formula (ZaUj :: i) cZcj (where Z represents a hydrophilic amino acid residue independently selected from the group consisting of R, D, E and K, U means a hydrophobic amino acid residue independently selected from the group consisting of V, I, L, C, Y and F, a has an average value of 1 10 to 5, b has an average value of 3 to 20, c is 1-10, and d is 0-3 which, when mixed with a pharmaceutically acceptable phospholipid, forms a synthetic surfactant with a surfactant activity greater than the surfactant activity of the phospholipid alone. A polypeptide according to claim 1, characterized in that a is 1-3, b is 3-10, c is 3-6, and d is 1 or 2. A polypeptide according to claim 1 or 2, characterized in that Z is K. The polypeptide of claim 1 or 2, characterized in that Z is R. A polypeptide according to claim 1 or 2, characterized in that Z is D. A polypeptide according to any one of the preceding claims, characterized in that U is L. A polypeptide according to claim 1, characterized in that it has an amino acid sequence selected from the group consisting of: DLLLLDLLLLDLLLLDLLLLD, 30 RLLLLRLLLLRLLLLRLLLLR, RLLLLLLLLRLLLLLLLLRLL, RRLLLLLLLRRLLLLLLLRRL, RLLLLCLLLRLLLLCLLLR, RLLLLCLLLRLLLLCLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL The polypeptide of claim 7, characterized in that it has the formula RLLLLCLLLiRIjLIjIjCljLLR. A polypeptide according to any one of claims 1-8, characterized in that it is mixed with one or more pharmaceutically acceptable phospholipids to form a pulmonary surfactant with a surfactant activity greater than the surfactant activity of phospholipid alone. Process for the preparation of a therapeutic drug usable for the treatment of respiratory distress syndrome, characterized in that 10 mixes one or more pharmaceutically acceptable phospholipids with a therapeutically effective amount of a polypeptide according to any one of claims 1-8.